1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid using a bacterium of the Enterobacteriaceae family.
2. Brief Description of the Related Art
Bacterial toxin-antitoxin pairs are typically made up of a stable toxin protein that can cause cell death by disrupting an essential cellular process, coupled with a labile antitoxin protein that can bind to and block activity of the toxin.
YoeB and YefM are a known toxin-antitoxin pair. The YoeB protein is similar to the Txe protein and the YefM protein is similar to the Axe protein. Both the Txe-Axe toxin-antitoxin pair are encoded by a multidrug resistance episome isolated from Enterococcus faecium (Grady, R. and Hayes, F., Mol. Microbiol., 47(5); 1419-32 (2003)). YoeB recognizes and binds to a linear peptide sequence within YefM (Cherny, I. and Gazit, E., J. Biol. Chem. 279(9); 8252-61 (2004)). The YoeB toxin induces cleavage of translated mRNAs. YoeB can be activated by overproduction of the Lon protease, which is lethal for bacterial cells (Christensen S. K. et al, Mol. Microbiol., 51(6); 1705-17 (2004)). The YefM protein appears to lack secondary structure, and its native conformation is proposed to be unfolded. A linear recognition element which is recognized by YoeB was identified using peptide array technology (Cherny, I. and Gazit, E., J. Biol. Chem. 279(9); 8252-61 (2004)).
YafQ and DinJ are another known toxin-antitoxin pair, with DinJ (DNA damage inducible protein) being the antitoxin for the YafQ protein (Gerdes, K., J. Bacteriol., 182(3): 561-72 (2000); Christensen S. K. et al, Mol. Microbiol., 51(6); 1705-17 (2004)). It was shown that under the stringent conditions of growth arrest, the YafQ and DinJ pair is significantly upregulated along with several other toxin-antitoxin pairs (Chang, D. E. et al, Mol. Microbiol., 45(2): 289-306 (2002)).
The mazEF system encodes the MazE-MazF toxin-antitoxin pair, with MazF being the toxin that is counteracted by the MazE antitoxin (Aizenman, E. et al, Proc. Natl. Acad. Sci. USA, 93 (12); 6059-63 (1996)). The MazE-MazF system mediates the toxicity of guanosine 3′,5′-bispyrophosphate (rapid relA induction), which is associated with amino acid deprivation (Aizenman, E. et al, Proc. Natl. Acad. Sci. USA, 93 (12); 6059-63 (1996)); cell death caused by the antibiotics rifampicin, chloramphenicol, and spectinomycin (Sat, B. et al, J. Bacteriol., 183(6); 2041-5 (2001)); and the thymineless death (TLD) response to thymine starvation (Sat, B. et al, J. Bacteriol., 185(6); 1803-7 (2003)). The MazE antitoxin is subject to degradation by the ClpP-ClpA protease complex and exhibits a short (30 minute) half life, whereas the toxin, MazF, is much more stable. It has been shown that overproduction of MazE has no effect on the absence of MazF (Aizenman, E. et al, Proc. Natl. Acad. Sci. USA, 93 (12); 6059-63 (1996)). MazF exhibits sequence-specific ribonuclease activity toward single- or double-stranded RNA regions (Munoz-Gomez, et al, FEBS Lett., 567(2-3); 316-20 (2004)), and the resulting degradation of cellular MRNA causes global translation inhibition (Zhang, Y. et al, Mol. Cell, 12(4); 913-23 (2003)). MazF exhibits RNase activity toward tmRNA, and tmRNA is involved in the release of MazF-mediated cell growth inhibition (Christensen, S. K. et al, J. Mol. Biol., 332(4); 809-19 (2003)). MazF also stimulates DNA binding by MazE (Zhang, J. et al, J. Biol. Chem. 278(34); 32300-6 (2003)).
RelE is the toxin in the RelE-RelB toxin-antitoxin system (Gotfredsen, M. and Gerdes, K., Mol. Microbiol. 29(4), 1065-76 (1998)). RelE and RelB proteins exhibit a physical interaction, and the RelE protein physically interacts with ribosomes (Galvani, C. et al, J. Bacteriol., 183(8), 2700-3 (2001)). RelE inhibits protein translation by catalyzing cleavage of mRNA in the A site of the ribosome (Pedersen, K. et al, Cell 112(1), 131-40 (2003)). RelE is involved in regulation of cellular protein translation when nutrients are limited (Christensen, S. K. et al, Proc. Natl. Acad. Sci. USA, 98(25), 14328-33 (2001); Pedersen, K. et al, Cell 112(1), 131-40 (2003); Christensen, S. K. and Gerdes, K., Mol. Microbiol., 48(5), 1389-400 (2003)). When cells are starved of amino acids, Lon protease degrades RelB protein; degradation of RelB protein derepresses transcription of relBE operon; RelE toxin accumulates in excess compared with its RelB antitoxin; and this free RelE toxin causes translation inhibition (Christensen, S. K. et al, Proc. Natl. Acad. Sci. USA, 98(25), 14328-33 (2001). RelE-mediated translation inhibition is reported to cause reversible inhibition of cell growth (Pedersen, K. et al, Mol. Microbiol., 45(2); 501-10 (2002)).
YeeV is a member of novel family of toxin proteins, ectopic expression of which caused growth inhibition. Coexpression of the gene upstream of each of these toxins restored the growth rate to that of the uninduced strain (Brown, J. M. and Shaw, K. J., J. Bacteriol., 185 (22), 6600-6608 (2003)).
The ability of Escherichia coli cells to survive prolonged exposure to penicillin antibiotics, called high persistence (hip), is associated with mutations in the hipA gene. The hip operon consists of two genes, hipA and hipB. The hipA gene encodes the HipA toxin, whereas hipB encodes a DNA-binding protein that autoregulates expression of the hipBA operon and binds to HipA to nullify its toxic effects (Korch, S. B., Henderson, T. A., and Hill, T. M., Mol. Microbiol., 2003, 50(4):1199-1213). Bacterial populations produce persisters, cells that neither grow nor die in the presence of bactericidal agents, and thus exhibit multidrug tolerance (MDT). Deletion of the hipBA operon causes a sharp decrease in persisters in both stationary and biofilm populations. The hipA gene is thus the first validated persister-MDT gene (Keren, I. et al., J. Bacteriol., 2004, 186(24):8172-8180). It has been shown that mutations in the hipA gene of Escherichia coli K-12 greatly reduce the lethality of selective inhibition of peptidoglycan synthesis. These mutations reduce the lethality that accompanies either selective inhibition of DNA synthesis or heat shock of strains defective in htpR. In addition, the mutant alleles of hipA are responsible for a reversible cold-sensitive block in cell division and synthesis of macromolecules, particularly peptidoglycan (Scherrer, R. and Moyed, H. S., J. Bacteriol., 1988, 170(8):3321-3326). It has also been shown that overexpression of hipA produces an antibiotic tolerance phenotype under conditions that do not affect the growth rate of the organism. Overexpressing hipA probably decreases the period in which bacteria are susceptible to the antibiotics by temporarily affecting some aspect of chromosome replication or cell division (Falla, T. J. and Chopra I., Antimicrob Agents Chemother., 1998, 42(12):3282-3284).
But currently, there have been no reports of attenuation of expression of a gene encoding a toxin of bacterial toxin-antitoxin pairs for the purpose of producing of L-amino acids.